The lymphatic vasculature is essential for the maintenance of fluid balance in the body, for immune defense and for the uptake of dietary fat. Absent or damage lymphatic vessels lead to lymphedema, a chronic and disfiguring swelling of the extremities. In cancer patients the lymphatic vessels serve as a major route for the spread of tumor cells. Our long-term goal is to understand, at the molecular level, the mechanisms of lymphatic vessel growth, to identify the key molecules in the lymphatic endothelial differentiation program, and to use this knowledge to generate and differentiate lymphatic endothelial cells in vitro with a view to potential clinical applications, such as treatment of lymphedema. To address the regulation of the lymphatic endothelial differentiation program, transcription factors specifically expressed in the lymphatic endothelial cells (LECs), such as Prox-1 and eight other novel transcription factors identified in our preliminary studies, LECs or they will be overexpressed in blood vascular endothelial cells (BECs), and changes in the transcriptional programs of the two cell types will be monitored using DNA microarrays. To confirm our results in vivo, transgenic mice expressing inducible Prox-1 in blood vessels will be produced, and the expression of potential Prox-1 target genes will be studied. The transcriptional programs initiated in BECs and LECs upon stimulation with major regulators of angiogenesis and lymphangiogenesis, VEGF and VEGF-C, respectively, as well as with ligands specific for the three known VEGF receptors, will be studied. To understand the differences between lymphatic endothelial from different organs, human intestinal and skin LECs will be isolated, and molecules differentially expressed in the two cell populations will be identified. The tissue-specific expression of cell surface proteins in LECs from the skin and gut will be investigated using the in vivo/in vitro phage peptide and cDNA display approach. These studies will provide fundamental new insights into the mechanisms of lymphatic endothelial growth and differentiation. They should also identify molecular markers distinguishing superficial and visceral lymphatic vessels, which could then be used to develop targeted drug delivery to these vessels.